In this application for a Fogarty International Research Collaboration Award we are proposing to establish collaborative approaches to address the problem of how retroviral protease activation is controlled prior to virus assembly and maturation. In addition, the specificity and role of the viral protease in mediating maturation of the viral capsid and glycoprotein will be studied. The laboratory of the PI has established, through molecular genetic approaches, a research program studying the assembly and replication of the primate D-type retrovirus, Mason-Pfizer Monkey Virus (M-PMV). The laboratory of Drs Strop and Sedlacek has expertise in the expression and analysis of cellular aspartyl proteinases and more recently has turned its attention to a similar class of enzymes found in retroviruses. Their established abilities to express such enzymes to high levels in bacteria, has enabled them to produce sufficiently large amounts of the avian myeloblastosis virus proteinase for structural studies. Moreover, the ability to produce large amounts of enzyme facilitated a detailed characterization of the proteinase and the development of novel inhibitors for this class of enzymes. These areas of expertise, which are not available in my research group, provide a unique opportunity for establishing a collaboration to address the problems described in this application. Specifically we propose to: 1. Develop an efficient heterologous expression system for the M-PMV proteinase in E. coli with the purpose of producing milligram amounts of protein for structural and functional studies. 2. Characterize the specificity of the M-PMV proteinase using synthetic substrates and inhibitors. New substrates corresponding to cleavage sites within the M-PMV precursor proteins as well as substrates and inhibitors previously characterized for other retroviral proteinase will be employed. 3. Investigate the processing of viral precursor proteins during assembly and maturation by utilizing inhibitors with different binding constants for in vitro and in vivo analyses. 4. Investigate the structure of both the active proteinase molecule(s) and inactive protease precursor by x-ray crystallography (in collaboration with Dr. Ming Luo, UAB). 5. Investigate in vitro the conditions for activation of proteinase in preassembled virions and for cleavage of the cytoplasmic domain of the viral TM protein (gp22).